A rapid detection method for genetic purity of BT-type cytoplasmic male sterile rice seeds based on real-time fluorescent quantitative PCR

Abstract

This application discloses a rapid detection method for the genetic purity of BT-type cytoplasmic male sterile rice seeds based on real-time quantitative PCR. The method primarily utilizes specific primers designed targeting sequences unique to maintainer lines in the rice mitochondrial genome, using the rice internal reference gene ATP6 as a reference to ensure high specificity and accuracy. A standard curve of a mixture of sterile and maintainer line seeds constructed based on this method allows the conversion of ΔCt values ​​into relative abundance for quantitative analysis. Results show that this method exhibits extremely strong linearity, accurate quantification in simulated mixed samples, and a near 100% recovery rate, providing a rapid, stable, and reliable detection method for the quality control of rice hybrids.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and agricultural science, specifically relating to a rapid detection method for the genetic purity of BT-type cytoplasmic male sterile rice seeds based on real-time quantitative PCR. Background Technology

[0002] Hybrid rice typically exhibits a significant yield advantage compared to traditional self-pollinating rice varieties, with yield increases reaching 20-30%. The commercial application of this advantage has not only significantly increased yield per unit area but also driven a substantial increase in total rice production. This demonstrates that high-purity seeds are a key prerequisite for ensuring that hybrid rice maintains its high-yield characteristics and achieves sustainable development.

[0003] To meet market demands, commercially available hybrid rice seeds must meet a minimum purity standard of 97%, while the purity requirement for parental lines used in three-line hybrid rice production is even higher, reaching 99.5%.

[0004] Taking the most widely used BT-type sterile line as an example, in production practice, cytoplasmic male sterile plants and their maintainer line seeds are the main reasons for the decline in the purity of hybrid seeds.

[0005] Therefore, it is urgent to strictly control the genetic purity of sterile seeds. The detection of seed genetic purity in related technologies mainly relies on field planting phenotypic identification. This method requires a complete growth cycle (4-6 months) and has significant drawbacks such as long detection cycle, high labor intensity, and susceptibility to environmental factors. It cannot meet the needs of modern seed industry for real-time monitoring of seed quality.

[0006] Therefore, in recent years, there have been reports of attempts to achieve rapid in vitro identification using molecular marker technologies, such as conventional PCR. However, as an endpoint detection technology, it can only provide qualitative results and cannot accurately quantify the contamination ratio, especially when the contamination rate is close to the standard threshold, making it difficult to make an accurate judgment. In addition, its process relies on gel electrophoresis, which is cumbersome, has low throughput, and poses a risk of cross-contamination.

[0007] In summary, this application provides a rapid detection method for the genetic purity of BT-type cytoplasmic male sterile rice seeds based on real-time quantitative PCR. This method is characterized by its speed, reliability, and high sensitivity, and can also achieve qualitative identification and precise quantitative analysis of maintainer line contamination in CMS seed batches. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides a rapid detection method for the genetic purity of BT-type cytoplasmic male sterile rice seeds based on real-time quantitative PCR. This method enables rapid qualitative identification and quantitative analysis of contamination of maintainer line seeds in BT-type CMS rice seeds.

[0009] In a first aspect, this application provides a rapid detection method for the genetic purity of BT-type cytoplasmic male sterile rice seeds based on real-time quantitative PCR, comprising the following steps:

[0010] 1) Extract genomic DNA from the rice seed samples to be tested;

[0011] 2) Using the DNA extracted in step 1) as a template, a real-time quantitative PCR system with SYBR Green fluorescent dye was used;

[0012] Then, amplification was performed using maintainer-specific primer pairs and internal reference gene primer pairs, respectively.

[0013] 3) Construct a standard curve and perform quantitative analysis on the maintainer seeds mixed in the sample.

[0014] Preferably, the sequence of the retention system-specific forward primer F is shown in SEQ ID No:1:

[0015] SEQ ID No.1 (5'-TCCCCACTAGTCTTCTTCGTACATG-3');

[0016] The sequence of the maintainer-specific reverse primer R is shown in SEQ ID No:2:

[0017] SEQ ID No.2 (5'-TGGCCCCTAAGAGGAAAAGC-3');

[0018] The maintainer-line specific primers were designed for the mitochondrial genomic differences between the BT-type cytoplasmic male sterile line and the maintainer line.

[0019] Preferably, the sequence of the forward primer F for the internal reference gene is shown in SEQ ID No:3:

[0020] SEQ ID No.3 (5'-GAATGGAGGAACGGCGATAG-3');

[0021] The sequence of the reverse primer R for the internal reference gene is shown in SEQ ID No:4:

[0022] SEQ ID No.4 (5'-CCGCCCTTTTTCGTAACAACT-3');

[0023] The internal reference gene primers were designed for the mitochondrial endogenous reference gene ATP6, which is conserved in both the BT-type cytoplasmic male sterile line and its corresponding maintainer line.

[0024] Preferably, the parameters for step (2) are as follows:

[0025] The real-time quantitative PCR system is 20 μL, containing:

[0026] 10 μL of 2× SYBR Green Master Mix, 0.4 μL each of 10 μM upstream and downstream primers, 2 μL of DNA template, and ddH2O to bring the total volume to 20 μL;

[0027] The quantitative analysis conditions were: 95℃ pre-denaturation for 20 seconds; 95℃ denaturation for 10 seconds; 60℃ annealing / extension for 30 seconds, for a total of 40 cycles.

[0028] Preferably, the method for constructing the standard curve in step 3) is as follows:

[0029] First, the genomic DNA of the maintainer line seeds and the genomic DNA of the sterile line seeds were mixed in different proportions, and then the ΔCt value was obtained by qPCR detection. The ΔCt value = internal reference gene Ct value - maintainer line specific gene Ct value. A linear regression equation between 2-ΔCt value and the mixing ratio of maintainer line seeds was established.

[0030] Secondly, this application provides a kit for detecting contamination of maintainer line seeds in BT-type cytoplasmic male sterile rice seeds, the kit comprising:

[0031] 1) Primer pairs used to amplify maintainer-specific sequences:

[0032] The sequence of the maintainer-specific forward primer F is shown in SEQ ID No:1:

[0033] SEQ ID No.1 (5'-TCCCCACTAGTCTTCTTCGTACATG-3');

[0034] The sequence of the maintainer-specific reverse primer R is shown in SEQ ID No:2:

[0035] SEQ ID No.2 (5'-TGGCCCCTAAGAGGAAAAGC-3');

[0036] 2) Primer pair used to amplify the internal reference gene ATP6:

[0037] The sequence of the forward primer F for the internal reference gene is shown in SEQ ID No:3:

[0038] SEQ ID No.3 (5'-GAATGGAGGAACGGCGATAG-3');

[0039] The sequence of the reverse primer R for the internal reference gene is shown in SEQ ID No:4:

[0040] SEQ ID No.4 (5'-CCGCCCTTTTTCGTAACAACT-3');

[0041] 3) SYBR Green fluorescent dye.

[0042] Preferably, the kit also contains 2×SYBR Green Master Mix, DNA extraction reagents and / or standards.

[0043] Thirdly, this application provides an application of any of the above methods or kits in the purity detection of BT-type cytoplasmic male sterile rice seeds, wherein the purity detection includes qualitative identification and quantitative analysis of the mixing ratio of maintainer line seeds in a seed batch, and is applied to the quality control of parent seeds (sterile lines) during hybrid rice seed production.

[0044] In summary, this application has the following beneficial effects:

[0045] 1) Compared with traditional field planting identification which takes 4-6 months, this application can complete the detection from sample to result in only 2-3 hours, which greatly shortens the detection cycle and meets the real-time monitoring needs of seed production, processing and sales.

[0046] 2) This application uses closed-tube operation throughout, which can effectively solve the problems of complicated operation and contamination. Using qPCR technology, amplification and detection are completed in closed tubes, eliminating the need for gel electrophoresis, avoiding cross-contamination, simplifying the operation process and reducing human error.

[0047] 3) This application can not only intuitively determine whether a sample is qualified through Ct value and standard curve, but also accurately calculate the mixing ratio, providing a quantitative basis for seed quality control with accuracy down to the numerical value. It has a decisive advantage, especially in judging whether the mixing ratio is close to the national standard critical value.

[0048] 4) The primers designed based on the mitochondrial-specific sequence of the maintainer line in this application, combined with the high sensitivity of quantitative fluorescence detection, can detect 0.1% or less of maintainer line seed contamination. The sensitivity is significantly higher than that of ordinary PCR, providing a reliable guarantee for the quality control of high-purity parental seeds.

[0049] 5) This application demonstrates, through the construction of standard curves and the validation of simulated contaminated samples, that the detection technology in this application has extremely high stability and accuracy (R²>0.99, spiked recovery rate close to 100%) among different operators and different batches, and is suitable for industrial and large-scale applications. Attached Figure Description

[0050] Figure 1This is a schematic diagram showing the results of specific detection of maintainer line seed-specific primers using conventional PCR in Example 1 of the present invention;

[0051] Where M: DNA molecular weight standard; NTC: blank control;

[0052] Figure 2 This is a schematic diagram showing the results of specific detection of the endogenous reference gene ATP6 primers using conventional PCR in Example 1 of the present invention;

[0053] Where M: DNA molecular weight standard; NTC: blank control;

[0054] Figure 3 This is a qPCR amplification curve of the endogenous reference gene in Example 1 of this invention;

[0055] Wherein, NTC: blank control;

[0056] Figure 4 This is a qPCR amplification curve of the seed-specific sequence of the maintainer line in Example 1 of the present invention;

[0057] NTC: Blank control.

[0058] Figure 5 In Embodiment 1 of this invention, the contamination ratio of the maintenance line seeds is compared with the corresponding 2 -ΔCt The standard curve generated by plotting the values;

[0059] ΔCt is defined as the average Ct value of the endogenous reference gene minus the average Ct value of the seed-specific sequence of the maintainer line. Detailed Implementation

[0060] The following is in conjunction with the appendix Figure 1-5 The present application will be further described in detail with reference to the embodiments.

[0061] Example 1

[0062] A rapid method for detecting the genetic purity of BT-type cytoplasmic male sterile rice seeds based on real-time quantitative PCR includes the following steps:

[0063] 1) Extract genomic DNA from the rice seed samples to be tested, as follows:

[0064] 1.1. Material Preparation - Seeds air-dried to a moisture content of 13±0.5%.

[0065] First, seeds of BT-type cytoplasmic male sterile lines (Shenchang A, Shen21A, Shen22A, Shen24A and Hanfeng A), corresponding maintainer line seeds (Shenchang B, Shen21B, Shen22B, Shen24B and Hanfeng B), and restorer line seed Shenhui 26 were selected for storage and analysis.

[0066] Seeds of the BT-type sterile line Shen 21A and its corresponding maintainer line Shen 21B were dehulled, and then the rice grains were ground into fine powder using an FW100 high-speed grinder from Tianjin Tairun Technology Instrument Co., Ltd. The resulting rice powders of Shen 21A and Shen 21B were thoroughly mixed in different proportions (5%, 2%, 1%, 0.5%, and 0.1%) to simulate different levels of seed contamination samples for constructing a standard curve.

[0067] 1.2. DNA template extraction

[0068] DNA template for primer specificity assay: Rice leaves were ground and DNA was extracted using the CTAB method. The concentration of the extracted DNA was determined using a Thermo Fisher Scientific NanoDrop 2000 spectrophotometer and adjusted to 10 ng / µl for later use.

[0069] DNA templates for standard curve construction: Sterile line seeds and maintainer line seeds were ground into powder using a grinder, mixed evenly according to the different proportions mentioned above, and genomic DNA was extracted using a DNA extraction kit for edible starch and its products. The specific procedures are as follows:

[0070] Transfer 100 mg of rice flour to a 2 mL microcentrifuge tube containing 800 μL of STS buffer and 20 μL of proteinase K. Vortex thoroughly to completely suspend the precipitate and incubate at 70 °C for 10 minutes. After centrifugation at 12,000 rpm for 5 minutes, transfer 300 μL of the supernatant to a new 2 mL centrifuge tube.

[0071] Next, add 600 μL of SWB buffer and 30 μL of starch-binding magnetic beads, and vortex for 1 minute. Incubate the mixture at room temperature for 10 minutes, vortexing for 1 minute every 3 minutes to promote DNA binding. Place the centrifuge tube on a magnetic separator for approximately 30 seconds until the magnetic beads are completely separated, and remove the supernatant. Wash the magnetic beads once with 600 μL of WB buffer and twice with 600 μL of SPW buffer. After incubating at room temperature for 5–10 minutes, elute the DNA with 50 μL of TE buffer. Use a NanoDrop 2000 spectrophotometer to determine the mass and concentration of the DNA solution. Once the DNA OD260 / 280 ratio is confirmed to be between 1.8 and 1.9, adjust the DNA solution concentration to 10 ng / µl for later use.

[0072] 2) Using the DNA extracted in step 1) as a template, amplification was performed using a real-time quantitative PCR system with SYBR Green fluorescent dye. Amplification was then carried out using maintainer-specific primer pairs and internal control gene primer pairs, respectively. The specific operations, methods, and preparation conditions are as follows:

[0073] 2.1. Primer Design and Screening

[0074] To identify rice mitochondrial-specific sequences, Nipponbare was selected as a representative maintainer line for all CMS types. The mitochondrial genomes of Nipponbare and BT-type CMS rice were compared and analyzed to identify mitochondrial sequences unique to Nipponbare. Based on these sequences, corresponding specific primers were designed. The primer sequences are as follows:

[0075] Maintainer-specific primers:

[0076] Forward primer F: SEQ ID No. 1 (5'-TCCCCACTAGTCTTCTTCGTACATG-3');

[0077] Reverse primer R: SEQ ID No.2 (5'-TGGCCCCTAAGAGGAAAAGC-3').

[0078] The mitochondrial ATP6 gene, which is conserved in both the BT-type CMS line and its corresponding maintainer lines, was selected as the endogenous reference gene. The primer sequences for ATP6 were designed as follows:

[0079] Forward primer F: SEQ ID No.3 (5'-GAATGGAGGAACGGCGATAG-3');

[0080] Reverse primer R: SEQ ID No.4 (5'-CCGCCCTTTTTCGTAACAACT-3').

[0081] 2.2. Verification of the specificity of conventional PCR primers

[0082] To verify the specificity of the primers designed in section 2.1, total genomic DNA from five BT-type sterile lines (Shenchang A, Shen21A, Shen22A, Shen24A, and Hanfeng A), their corresponding maintainer lines (Shenchang B, Shen21B, Shen22B, Shen24B, and Hanfeng B), and the restorer line Shenhui 26 was used as templates for conventional PCR amplification. The conventional PCR reaction system is as follows:

[0083] The total volume is 20 μL, which also includes: 20 ng DNA template, 10 μL 2× Rapid Taq Master Mix (P222, Nanjing Novizan Biotechnology Co., Ltd.), and upstream and downstream primers with a final concentration of 0.25 μM.

[0084] The PCR reaction procedure was as follows: 95℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 35 cycles; and a final extension at 72℃ for 5 min. After amplification, the PCR products were separated by electrophoresis on a 2% (w / v) agarose gel, and the results were observed by UV imaging.

[0085] The results are as follows Figure 1 As shown, when the maintainer-specific primers (SEQ ID No: 1-2) were used, amplification products of approximately 200 bp were detected in both the maintainer and restorer lines, while no amplification products were observed in the sterile line or the blank control (NTC).

[0086] from Figure 2 It can be seen that when using the endogenous reference gene ATP6 primer (SEQ ID No:3-4), a single specific amplification product of about 200 bp in size can be detected in the sterile line, maintainer line and restorer line samples, while no product is detected in NTC.

[0087] The above results confirm that the maintainer-specific primers have high specificity, and also verify that ATP6 can be used as an endogenous reference gene for subsequent quantitative analysis.

[0088] 2.3. Real-time quantitative PCR detection

[0089] Using the DNA extracted in step 1.2 as a template, and employing the maintainer-line specific primers (SEQ ID No: 1-2) and the internal reference gene ATP6 primers (SEQ ID No: 3-4) designed in step 2.1, a real-time quantitative PCR reaction system based on SYBR Green fluorescent dye was established, as follows:

[0090] Reaction system (20 μL):

[0091] 2× SYBR Green Master Mix (QPX-201, Toyobo (Shanghai) Biotechnology Co., Ltd.): 10μL;

[0092] Upstream primer (10 μM): 0.4 μL;

[0093] Downstream primer (10 μM): 0.4 μL;

[0094] DNA template: 2 μL;

[0095] ddH2O: Add to a final volume of 20 μL;

[0096] Reaction procedure:

[0097] Pre-denaturation at 95℃ for 20 seconds;

[0098] Denaturation at 95℃: 10s;

[0099] Annealing / extending at 60℃: 30s, 40 cycles in total;

[0100] Each sample was tested in triplicate, with homozygous Shen21B seeds and a blank control (NTC) serving as positive and negative controls, respectively. qPCR was performed using maintainer-specific primers and the internal reference gene ATP6 primers, respectively. The results are shown below. Figure 3 and Figure 4 As shown, the specific analysis is as follows:

[0101] Figure 3 The use of ATP6 primers produced highly similar amplification curves in CMS-maintainer mixed samples, homozygous maintainer samples, and blank controls with different proportions. The corresponding Ct values ​​showed minimal variation among different samples, indicating that the ATP6 gene amplification stability was good.

[0102] from Figure 4 As can be seen, when using maintainer-specific primers to amplify CMS-maintainer mixed samples with different proportions, the Ct value decreases in a gradient as the proportion of maintainer seed increases, and is negatively correlated with the abundance of target sequences in the sample.

[0103] 3) Construct a standard curve using the mixed sample obtained in 1.1, and perform quantitative analysis on the maintainer seeds mixed in the sample. The specific operation method is as follows:

[0104] 3.1. Construction of the Standard Curve

[0105] Seed samples of BT-type CMS line Shen21A and its corresponding maintainer line Shen21B with different proportions (5%, 2%, 1%, 0.5% and 0.1%) obtained in 1.1 were selected, and genomic DNA was extracted from each sample and then subjected to quantitative analysis and standardization.

[0106] Then, operators A and B independently constructed four independent experimental batches at two different time periods. Each batch contained five mixed samples in different proportions. Each sample was measured three times in total. Real-time quantitative PCR was then performed. For each reaction, the ΔCt value was the difference between the Ct value of the maintainer line-specific target gene and the internal reference gene ATP6, i.e., ΔCt = Ct(ATP6).

[0107] -Ct (maintainer line specific gene), the Ct and ΔCt values ​​of each proportion of samples are recorded in Table 1.

[0108] Through 2 -ΔCt The conversion method transforms the average ΔCt values ​​obtained from each group of experiments into a 2... -ΔCtThe x-axis represents the values, and the y-axis represents the corresponding percentage of contaminated seeds in the maintainer lines. A standard curve is plotted, referencing... Figure 5 The linear regression equation of the standard curve is y = 1.2005x + 0.0502, R² = 0.9986, indicating that the quantitative results are accurate and reliable.

[0109] 3.2. Validation using simulated contaminated samples

[0110] To verify the accuracy of the established qPCR method in quantifying low-level seed contamination, simulated samples containing 0.2%, 1.5%, and 2.5% maintainer line contamination were prepared. Genomic DNA was extracted from each simulated sample, quantified uniformly, and subjected to real-time PCR based on SYBR Green.

[0111] Each simulated sample was repeated three times. Then, based on the standard curve constructed in step 3.1, the corresponding contamination ratio of the maintainer seed was estimated by interpolation. The spiked recovery rate was calculated as the ratio of the estimated contamination ratio to the nominal contamination ratio, expressed as a percentage, and recorded in Table 2.

[0112]

[0113] As shown in Table 2, the measured values ​​of pollution at each detection concentration were highly consistent with the preset values, and the spiked recovery rate ranged from 93.27% to 109.18%, with an average spiked recovery rate close to 100%. This indicates that the method has high analytical accuracy and good repeatability within the range of pollutants detected.

[0114] Furthermore, data from the 0.2% simulated sample group show that even at a low contamination level of 0.2%, this method can reliably and accurately quantify maintainer line contamination in CMS seed batches.

[0115] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A rapid method for detecting the genetic purity of BT-type cytoplasmic male sterile rice seeds based on real-time quantitative PCR, characterized in that, Includes the following steps: 1) Extract genomic DNA from the rice seed samples to be tested; 2) Using the DNA extracted in step 1) as a template, a real-time quantitative PCR system with SYBR Green fluorescent dye was used; Then, amplification was performed using maintainer-specific primer pairs and internal reference gene primer pairs, respectively. 3) Construct a standard curve and perform quantitative analysis on the maintainer seeds mixed in the sample.

2. The rapid detection method for genetic purity of BT-type cytoplasmic male sterile rice seeds based on real-time quantitative PCR according to claim 1, characterized in that: The sequence of the maintainer-specific forward primer F is shown in SEQ ID No:1: SEQ ID No.1 (5'-TCCCCACTAGTCTTCTTCGTACATG-3'); The sequence of the maintainer-specific reverse primer R is shown in SEQ ID No:2: SEQ ID No.2 (5'-TGGCCCCTAAGAGGAAAAGC-3'); The maintainer-line specific primers were designed for the mitochondrial genomic differences between the BT-type cytoplasmic male sterile line and the maintainer line.

3. The rapid detection method for genetic purity of BT-type cytoplasmic male sterile rice seeds based on real-time quantitative PCR according to claim 2, characterized in that: The sequence of the forward primer F for the internal reference gene is shown in SEQ ID No:3: SEQ ID No.3 (5'-GAATGGAGGAACGGCGATAG-3'); The sequence of the reverse primer R for the internal reference gene is shown in SEQ ID No:4: SEQ ID No.4 (5'-CCGCCCTTTTTCGTAACAACT-3'); The internal reference gene primers were designed for the mitochondrial endogenous reference gene ATP6, which is conserved in both the BT-type cytoplasmic male sterile line and its corresponding maintainer line.

4. The rapid detection method for genetic purity of BT-type cytoplasmic male sterile rice seeds based on real-time quantitative PCR according to claim 1, characterized in that, The parameters for step (2) are as follows: The real-time quantitative PCR system is 20 μL, containing: 10 μL of 2× SYBR Green Master Mix, 0.4 μL each of 10 μM upstream and downstream primers, 2 μL of DNA template, and ddH2O to bring the total volume to 20 μL; The quantitative analysis conditions were: 95℃ pre-denaturation for 20 seconds; 95℃ denaturation for 10 seconds; 60℃ annealing / extension for 30 seconds, for a total of 40 cycles.

5. The rapid detection method for genetic purity of BT-type cytoplasmic male sterile rice seeds based on real-time quantitative PCR according to claim 4, characterized in that, The method for constructing the standard curve in step 3) is as follows: First, maintainer line seed genomic DNA and male-sterile line seed genomic DNA were mixed in different proportions, and then the ΔCt value was obtained by qPCR detection. The ΔCt value = internal reference gene Ct value - maintainer line specific gene Ct value. A 2-cell genomic DNA model was then established. -ΔCt The linear regression equation between the value and the proportion of seed mixing in the maintainer line.

6. A kit for detecting contamination of maintainer line seeds in BT-type cytoplasmic male sterile rice seeds, characterized in that, The kit contains: 1) Primer pairs used to amplify maintainer-specific sequences: The sequence of the maintainer-specific forward primer F is shown in SEQ ID No:1: SEQ ID No.1 (5'-TCCCCACTAGTCTTCTTCGTACATG-3'); The sequence of the maintainer-specific reverse primer R is shown in SEQ ID No:2: SEQ ID No.2 (5'-TGGCCCCTAAGAGGAAAAGC-3'); 2) Primer pair used to amplify the internal reference gene ATP6: The sequence of the forward primer F for the internal reference gene is shown in SEQ ID No:3: SEQ ID No.3 (5'-GAATGGAGGAACGGCGATAG-3'); The sequence of the reverse primer R for the internal reference gene is shown in SEQ ID No:4: SEQ ID No.4 (5'-CCGCCCTTTTTCGTAACAACT-3'); 3) SYBR Green fluorescent dye.

7. The kit for detecting contamination of maintainer line seeds in BT-type cytoplasmic male sterile rice seeds according to claim 6, characterized in that, The kit also includes 2×SYBR Green Master Mix, DNA extraction reagents and / or standards.

8. The application of the method according to any one of claims 1-5 or the kit according to any one of claims 6-7 in the purity detection of BT-type cytoplasmic male sterile rice seeds.

9. The application according to claim 8, characterized in that, The purity test includes qualitative identification and quantitative analysis of the proportion of maintainer seeds mixed in a seed batch.

10. The application according to claim 8, characterized in that, The application is to control the quality of parent seeds (sterile lines) during the hybrid rice seed production process.

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